Formulation and use of carotenoids in treatment of cancer

ABSTRACT

A reduced-toxicity formulation of carotenoids is disclosed which is stable in an aqueous environment. The formulation includes a carotenoid, lipid carrier particles (such as liposomes), and an intercalation promoter agent (such as a triglyceride), which causes the carotenoid to be substantially uniformly distributed with the lipid in the lipid carrier particles. The molar ratio of carotenoid to lipid is greater than about 1:10. Also disclosed is a method of inhibiting the growth of cancer cells, which comprises administering to a living subject a therapeutically effective amount of a composition as described above.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 08/286,928, filed onAug. 8, 1994, now aban. which is a continuation-in-part of U.S. Ser. No.08/213,249, filed on Mar. 14, 1994 now abandoned, which is acontinuation of U.S. Ser. No. 07/822,055, filed on Jan. 16, 1992, nowabandoned, which is a continuation-in-part of U.S. Ser. No. 07/588,143,filed on Sep. 25, 1990, now abandoned, which is a divisional of U.S.Ser. No. 07/152,183, filed on Feb. 4, 1988, now abandoned. The 152,183application is also a continuation in part of U.S. Ser. No. 051,890,filed on May 19, 1987, issued as U.S. Pat. No. 4,863,739, Sep. 5, 1989.The above-identified applications are incorporated here by reference.

BACKGROUND OF THE INVENTION

The present invention relates to therapeutic compositions of carotenoidsencapsulated in liposomes or other lipid carrier particles.

It has been known for more than 50 years that retinoids, the family ofmolecules comprising both the natural and synthetic analogues of retinol(vitamin A), are potent agents for control of both cellulardifferentiation and cellular proliferation (Wolbach et al., J. Exp.Med., 42:753-777, 1925). Several studies have shown that retinoids cansuppress the process of carcinogenesis in vivo in experimental animals(for reviews, see e.g., Bollag, Cancer Chemother. Pharmacol., 3:207-215,1979, and Sporn et al., In Zedeck et al. (eds.), Inhibition of Tumorinduction and development, pp. 71-100. New York: Plenum PublishingCorp., 1981). These results are now the basis of current attempts to useretinoids for cancer prevention in humans. Furthermore, there isextensive evidence which suggests that retinoids can suppress thedevelopment of malignant phenotype in vitro (for review, see e.g.,Bertram et al., In: M. S. Arnott et al., (eds.), Molecular interactionsof nutrition and cancer, pp 315-335. New York, Raven Press, 1982; Lotanet al., The modulation and mediation of cancer by vitamins, pp 211-223.Basel: S. Karger AG, 1983) thus suggesting a potential use of retinoidsin cancer prevention. Also, recently it has been shown that retinoidscan exert effects on certain fully transformed, invasive, neoplasticcells leading in certain instances to a suppression of proliferation(Lotan, Biochim. Biophys. Acta, 605:33-91, 1980) and in other instancesto terminal differentiation of these cells, resulting in a more benign,non-neoplastic phenotype (see e.g., Brietman et al., Proc. Natl. Acad.Sci. U.S.A., 77:2936-2940, 1980).

Retinoids have also been shown to be effective in the treatment ofcystic acne (see e.g., Peck, et al., New Engl. J. Med., 300:329-333,1979). In addition to cystic acne, retinoid therapy has been shown to beeffective in gram-negative folliculitis, acne fulminans, acneconglobata, hidradenitis suppurativa, dissecting cellulitis of thescalp, and acne rosacea (see e.g., Plewig et al., J. Am. Acad.Dermatol., 6:766-785, 1982).

However, due to highly toxic side effects of naturally occurring formsof vitamin A (hypervitaminosis A) at therapeutic dose level, clinicaluse of retinoids has been limited (Kamm et al., In: The Retinoids. Spornet al., (eds.), Academic Press, N.Y., pp 228-326, 1984; Lippman et al.,Cancer Treatment Reports, 71:493-515, 1987). In free form, the retinoidsmay have access to the surrounding normal tissues which might be thebasis of their profound toxicity to liver, central nervous system, andskeletal tissue.

Therefore, one potential method to reduce the toxicity associated withretinoid administration would be the use of a drug delivery system. Theliposomal format is a useful one for controlling the topography of drugdistribution in vivo. This, in essence, involves attaining a highconcentration and/or long duration of drug action at a target (e.g. atumor) site where beneficial effects may occur, while maintaining a lowconcentration and/or reduced duration at other sites where adverse sideeffects may occur (Juliano, et al., In: Drug Delivery Systems, Julianoed., Oxford Press, N.Y., pp 189-230, 1980). Liposome-encapsulation ofdrug may be expected to impact upon all the problems of controlled drugdelivery since encapsulation radically alters the pharmacokinetics,distribution and metabolism of drugs.

There are additional difficulties in using a liposomal formulation of aretinoid for therapeutic purposes. For example, it is often desirable tostore the composition in the form of a preliposomal powder, but manyprior formulations are not satisfactory for such use, because theyeither contain an inadequate amount of retinoid, or they generateundesirable liposomes when they are reconstituted in aqueous solution.

For compositions that are to be administered intravenously, typicallythe composition must provide at least about 100 mg of the activeingredient in a single container; if it contains a lesser amount of theactive ingredient, an impractically large number of vials will be neededfor dosing a single patient.

Typically a vial having a volume of 120 cc is the largest that can beaccommodated in a commercial freeze drier, and 50 cc is the maximumvolume of liquid that can be filled in such a vial. If more than 1 g oflipids are included in 50 cc of liquid volume, the resulting liposomesafter reconstitution have a size distribution which is not acceptablefor parenteral administration. This is because the packing of the lipidsduring lyophilization is affected by the concentration of the lipids inthe solution. Thus, the concentration of lipids in the solution must belimited. However, when this is done in previously-known liposomalretinoid formulations, the retinoid tends to crystallize, and separatefrom the liposomes shortly after reconstitution.

In order to both limit the concentration of lipids and supply asufficient amount of retinoid, it is necessary to provide a molar ratioof retinoid to lipid greater than about 1 to 10. Previously knownformulations have not had, and are believed not to be capable of havingsuch a high packing of retinoid in the liposomes. Therefore, a needexists for improved compositions and methods which will minimize oreliminate the problems of the prior art.

SUMMARY OF THE INVENTION

The present invention relates to therapeutically useful, reducedtoxicity compositions of carotenoids. The compositions comprise acarotenoid, lipid carrier particles, and an intercalation promoteragent. "Carotenoid" is used here to include retinoids, pro-retinoids,carotenes, xanthophylls, and analogs thereof. A preferred example isall-trans retinoic acid. The carotenoid is substantially uniformlydistributed with the lipid in the lipid carrier particles. Moreparticularly, the carotenoid is substantially uniformly distributed inan intercalated position throughout a hydrophobic portion of the lipidcarrier particles, as opposed to the aqueous phase. "Substantiallyuniformly distributed" means that at least 50% of the lipid carrierparticles will contain carotenoid in a molar ratio between about 5:85carotenoid:lipid and about 15:70. Preferably at least 75% of all lipidcarrier particles will contain such a ratio of the active ingredient.

The composition is stable in an aqueous environment. In this context,"stable in an aqueous environment" means that the composition (1) willnot exhibit any therapeutically significant degradation over a period ofat least 24 hours, (2) will not exhibit a substantial degree of fusionsof liposomes over that same period, and (3) will not exhibit substantialredistribution of the carotenoid over that same period, including nosubstantial movement of the drug into the aqueous phase of a liposome,and no substantial state change into a crystalline form.

The molar ratio of carotenoid to lipid in the lipid carrier particles isgreater than about 1:10, and is most preferably at least about 15:85.The intercalation promoter agent preferably comprises at least about 15%by weight of the composition, and can suitably be, for example, atriglyceride.

"Lipid carrier particles" is used here to include liposomes, having abilayer structure formed of one or more lipids having polar heads andnonpolar tails, as well as micelles, amorphous particulates of lipid,and other lipid emulsion state entities. When the particles areliposomes, suitable forms include multilamellar liposomes.

The present invention also relates to a pharmaceutical unit dosageformulation of a carotenoid, which comprises a carotenoid, lipid carrierparticles, an intercalation promoter agent, and a pharmaceuticallyacceptable carrier. As stated above, the carotenoid is substantiallyuniformly distributed with the lipid in the lipid carrier particles, andthe composition is stable in an aqueous environment.

In another aspect, the invention relates to a method of inhibiting thegrowth of cancer cells, in which a therapeutically effective amount of acarotenoid composition is administered to a living subject. Thecarotenoid composition can be as described above. The composition ispreferably administered to the subject in a maintained molar ratiobetween about 5:85 carotenoid:lipid and about 15:70. "Maintained" inthis context means that the stated ratio of drug to lipid lasts for atleast 24 hours.

The present invention provides the therapeutic benefits of thecarotenoid, while substantially reducing the undesirable toxicity of thecomposition, as compared to the free drug. For example, encapsulation ofretinoic acid in liposomes results in a decrease of at least 15-fold intoxicity as compared to the free drug.

Further, the presence of the intercalation promoter agent permits theratio of active ingredient to lipid to be increased above what has beenpreviously known, and thus makes such formulations useful in a practicalsense for lyophilization into a powder, and subsequent reconstitutioninto solution which can be administered parenterally to a patient.Without wishing to be bound by any particular theory, it is believedthat the intercalation promoter agent overcomes steric hindrance thatotherwise limits the amount of carotenoid that be incorporated in, forexample, a liposome.

The encapsulation of carotenoids within, e.g., liposomes, permits theirdirect delivery to intracellular sites and thus circumvents therequirement for cell surface receptors. This may be of particularsignificance, for example, in therapy of tumors which lack the cellsurface receptors for serum retinol binding protein but possessintracellular receptors for retinoic acid.

Compositions of the present invention are also substantially improvedover prior liposomal retinoid formulations in terms of uniformity ofdrug distribution. Prior compositions often had substantial percentagesof liposomes which contained essentially no drug. In the presentinvention, at least 50% and preferably at least 75% of all liposomes inthe composition contain drug with the range specified above.

While not being bound by any particular theory of action, it has beenfound that, surprisingly, liposome encapsulation of carotenoids andparticularly all-trans retinoic acid, circumvents the usual hepaticclearance mechanisms. This has resulted in a substantial extension ofthe efficacy of liposomal carotenoid over free carotenoid or retinoid.It is believed that liposomal all-trans retinoic acid avoids theproblems of resistance to non-liposomal all-trans retinoic acid. Thisresistance is displayed by such parameters as reduced serumconcentration upon prolonged treatment typically observed in treatmentsas extended over 2, 5 or 7 weeks or longer. Here, substantially longerperiods of drug administration were unaccompanied by reduced circulatingdrug levels. Therapeutic i.v. dosages of 15 mg/m², 30 mg/m², 60 mg/m²,75 mg/m², and 90 mg/m², and further including 150 mg/m², 300 mg/m² andhigher are noted. Regimens of therapy extending in excess of 7 weeks,and further in excess of 14 weeks, and further exhibiting non-decliningdrug levels are particularly noted. Regimens of administration ofall-trans retinoic acid that avoid retinoid resistance are particularlynoted herein, which includes administration of liposomal all-transretinoic acid, and in one embodiment includes the retinoid beingintercalated in the liposomal bilayer in substantially uncrystallizedform.

In vivo administration of liposomal all-trans retinoic acid over aprolonged period did not exhibit declining blood levels. In vitrostudies of isolated liver microsomes revealed unchanged catabolism uponrepeated exposure to liposomal all-trans retinoic acid. In contrast,microsomes isolated from subjects originally administered non-liposomalall-trans retinoic acid an equal number of times displayed increasedmetabolism of all-trans retinoic acid.

Test data has indicated that the instant liposomal carotenoidformulations avoid "retinoid" resistance upon chronic i.v.administration. The results suggest that chronic administration ofliposomal carotenoid, and particularly all-trans retinoic acid, does notaffect the levels of circulating drug in subjects. While phospholipidsare preferred, the broad grouping of lipids are useful in formingparticular liposomes. Liver microsomes from test animals did not showany significant change in their ability to metabolize all-trans retinoicacid. In contrast, long-term oral administration of non-liposomalall-trans retinoic acid caused a significant decrease in circulatingdrug levels after 7 weeks of treatment. Liver microsomes from theseanimals converted all-trans retinoic acid into polar products much morerapidly than microsomes obtained from liposomal all-trans retinoicacid-treated or untreated animals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a time profile of liposomal retinoic acid (L-RA) stabilityin the presence () and absence (◯) of serum.

FIG. 2 shows human red blood cell (RBC) lysis as a function of time withRA () and L-RA (▴).

FIG. 3 shows RBC lysis as a function of retinoic acid (RA) concentration() and L-RA concentration (▴).

FIG. 4 shows the inhibition of THP-1 cell growth as a function of RAconcentration () , L-RA concentration (◯) or empty liposomeconcentration (Δ).

FIG. 5 shows the induction of transglutaminase (TGase) in humanmonocytic THP-1 cells as a function of treatment with RA or L-RA.

FIG. 6 shows the inhibition of human histiocytic U-937 cell growth as afunction of RA concentration (), L-RA concentration (◯) and emptyliposome concentration (Δ).

FIG. 7 shows the time course of accumulation of tissue TGase activity incultured human peripheral blood monocytes (HPBM). HPBM were fractionatedinto small (◯) and large () subpopulations by centrifugal elutriation,and they were cultured in 35-mm-well tissue culture plates as describedin Materials and Methods. At the indicated time points the cells werewashed, sonicated, and assayed for TGase activity. Values are the meansof six determinations from two dishes.

FIG. 8 shows dose-dependent effects of recombinant interferon-gamma(rIFN-g) on induction of tissue TGase activity in HPBM subpopulations.Small (◯) and large () monocytes were cultured in serum containingmedium alone or medium containing increasing concentrations of rIFN-g.After 72 hr, the cells were harvested and the cell lysates assayed fortissue TGase activity. The results shown represent mean ±SD of threedeterminations from an individual donor.

FIGS. 9A and B show effects of retinol (ROH) and RA on induction oftissue TGase activity in cultured HPBM. Cells were cultured in thepresence of 5% human AB serum and the absence () or presence of 500 nMROH (▴) or RA (◯) for varying periods of time. At the end of each timepoint, the cells were harvested and assayed for enzyme activity. Valuesshown are the means ±SD of six determinations from two independentexperiments. Inset, dose-response curve for tissue TGase induction byROH (▴) and RA () in HPBM after 72-hr culture.

FIGS. 10A and B show effects of free- and liposome-encapsulated RA oninduction of tissue TGase in HPBM. A: The cells were cultured in tissueculture dishes in presence of serum-containing medium alone (Δ) 500 nmliposomal RA (), or medium containing 500 nM free-RA (▴), or "emptyliposomes" (◯) for indicated periods of time. Both the liposomal RA and"empty liposomes" contained 200 μg/ml lipid. At the end of each timepoint, the cultures were washed and cell lysates assayed for TGaseactivity. Values shown are the mean ±SD of six determinations from twoindependent experiments. B: Western-blot analysis of the levels oftissue TGase in freshly isolated HPBM (lane 1) and in HPBM cultured for72 hr in the presence of serum-containing medium alone (lane 2), inmedium containing 500 nM free RA (lane 3), 500 nM liposomal RA (lane 4),or "empty liposomes" (lane 5). Cell lysates containing 25 ug of proteinwere subjected to Western-blot analysis as described in Materials andMethods.

FIGS. 11A and B show effect of free and liposome-encapsulated ROH oninduction of tissue TGase in HPBM. A: HPBM monolayers were cultured inserum-containing medium alone (Δ) or medium containing 1 μM of free- (◯)or liposomal-ROH (▴) for 72 hr. Then the cultures were washed and thecell lysates assayed for enzyme activity as described in Materials andMethods. B: Western-blot analysis of tissue TGase levels in freshlyisolated HPBM (lane 1) and in HPBM cultured for 72 hr in the presence ofserum-containing medium alone (lane 2), in medium containing 1 μM offree ROH (lane 3), or liposome-encapsulated ROH (lane 4) as described inMaterials and Methods. Twenty-five micrograms of cell protein was loadedonto each lane.

FIG. 12(A) shows the levels of all-trans retinoic acid in the blood 60min after oral administration of non-liposomal all-trans retinoic acidor i.v. administration of liposomal all-trans retinoic acid.

FIG. 12 (B) shows blood clearance of all-trans retinoic acid followingadministration of the last dose of all-trans retinoic acid.

FIG. 13(A) shows the percentage of all-trans retinoic acid metabolizedby isolated liver microsomes to animals exposed to 7 weeks of treatmentwith all-trans retinoic acid, either liposomal i.v. or oral.

FIG. 13(B) shows radioactivity (cpm) of all-trans retinoic acid or itspolar metabolites (as discussed in association with FIG. 12(A)) asgathered from five animals.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

Suitable therapeutic carotenoids for encapsulation in accordance withthe present invention include various retinoids. Trans-retinoic acid andall-trans-retinol are preferred. Other retinoids that are believedsuitable include: retinoic acid methyl ester, retinoic acid ethyl ester,phenyl analog of retinoic acid, etretinate, retinol, retinyl acetate,retinaldehyde, all-trans-retinoic acid, and 13-cis-retinoic acid.

Lipid carrier particles, such as liposomes, can be formed by methodsthat are well known in this field. Suitable phospholipid compoundsinclude phosphatidyl choline, phosphatidic acid, phosphatidyl serine,sphingolipids, sphingomyelin, cardiolipin, glycolipids, gangliosides,cerebrosides, phosphatides, sterols, and the like. More particularly,the phospholipids which can be used include dimyristoyl phosphatidylcholine, egg phosphatidyl choline, dilauryloyl phosphatidyl choline,dipalmitoyl phosphatidyl choline, distearoyl phosphatidyl choline,1-myristoyl-2-palmitoyl phosphatidyl choline, 1-palmitoyl-2-myristoylphosphatidyl choline, 1-palmitoyl-2-stearoyl phosphatidyl choline,1-stearoyl-2-palmitoyl phosphatidyl choline, dioleoyl phosphatidylcholine, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid,dimyristoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidylethanolamine, dimyristoyl phosphatidyl serine, dipalmitoyl phosphatidylserine, brain phosphatidyl serine, brain sphingomyelin, dipalmitoylsphingomyelin, and distearoyl sphingomyelin.

Phosphatidyl glycerol, more particularly dimyristoyl phosphatidylglycerol (DMPG), is not preferred for use in the present invention. Inthe carotenoid compositions of the present invention, the presence ofDMPG correlates with the appearance of amorphous structures of anomaloussize, which are believed to render the composition much less suitablefor intravenous administration. When DMPG is omitted, the amorphousstructures are not observed. The undesirable effects that are apparentlycaused by the presence of DMPG may result from the fact that DMPG has anegative charge, which may interact with the carboxylate of thecarotenoid.

In addition, other lipids such as steroids and cholesterol may beintermixed with the phospholipid components to confer certain desiredand known properties on the resultant liposomes. Further, syntheticphospholipids containing either altered aliphatic portions, such ashydroxyl groups, branched carbon chains, cyclo derivatives, aromaticderivatives, ethers, amides, polyunsaturated derivatives, halogenatedderivatives, or altered hydrophilic portions containing carbohydrate,glycol, phosphate, phosphonate, quaternary amine, sulfate, sulfonate,carboxy, amine, sulfhydryl, imidazole groups and combinations of suchgroups, can be either substituted or intermixed with the phospholipids,and others known to those skilled in the art.

A suitable intercalation promoter agent will permit the high molar ratioof carotenoid to lipid that is desired for the present invention,without substantial crystallization from the liposomes after they arereconstituted in aqueous solution, as can be observed by microscopicanalysis, separation techniques based on buoyant density, or othertechniques well known to those skilled in the art. Triglycerides arepreferred intercalation promoter agents, with soybean oil as onespecific example. Other suitable agents include sterols, such ascholesterol, fatty alcohols, fatty acids, fatty acids esterified to anumber of moieties, such as polysorbate, propylene glycol, mono- anddiglycerides, and polymers such as polyvinyl alcohols.

Prior to lyophilization, the carotenoid, lipids, and intercalationpromoter agent can be dissolved in an organic solvent, such ast-butanol. Lyophilization to form a preliposomal powder can be performedusing commercial apparatus which is known to persons skilled in thisfield. After lyophilization, the powder can be reconstituted as, e.g.,liposomes, by adding a pharmaceutically acceptable carrier, such assterile water, saline solution, or dextrose solution, with agitation,and optionally with the application of heat.

A preferred formulation, which can be dissolved in 45 ml of t-butanol,is as follows:

    ______________________________________                                        component  mg     millimoles   mole %                                                                              wt %                                     ______________________________________                                        DMPC       850    1.28         72    77                                       soybean oil                                                                              150    0.17         9     14                                       tretinoin  100    0.33         19    9                                        ______________________________________                                    

A composition of the present invention is preferably administered to apatient parenterally, for example by intravenous, intraarterial,intramuscular, intralymphatic, intraperitoneal, subcutaneous,intrapleural, or intrathecal injection. Administration could also be bytopical application or oral dosage. Preferred dosages are between 40-200mg/m². The dosage is preferably repeated on a timed schedule until tumorregression or disappearance has been achieved, and may be in conjunctionwith other forms of tumor therapy such as surgery, radiation, orchemotherapy with other agents.

The present invention is useful in the treatment of cancer, includingthe following specific examples: hematologic malignancies such asleukemia and lymphoma, carcinomas such as breast, lung, and colon, andsarcomas such as Kaposi's sarcoma.

EXAMPLE 1 Preparation of liposomal-all trans-retinoic acid (L-RA)

Preparation of lyophilized powder containing all trans-retinoic acid andphospholipids was carried out as follows. A solution of retinoic acid int-butanol (1-5 mg/ml) was added to a dry lipid film containingdimyristoyl phosphatidyl choline (DMPC) and dimyristoyl phosphatidylglycerol (DMPG) at a 7:3 molar ratio. The phospholipids were solubilizedin the t-butanol containing the all-trans retinoic acid and the solutionwas freeze-dried overnight. A powder containing dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidyl glycerol (DMPG), and all-transretinoic acid was obtained. The lipid:drug ratio used was from 10:1 to15:1.

Reconstitution of liposomal retinoic acid from the lyophilized powderwas done as follows. The lyophilized powder was mixed with normal salineat room temperature to form multilamellar liposomes containing alltrans-retinoic acid. This reconstitution method required mildhand-shaking for 1 min to obtain a preparation devoid of any aggregatesor clumps. By light microscopy, the reconstituted preparation containedmultilamellar liposomes of a close size range. No aggregates or drugclumps were identified in the liposomal preparation in three differentexperiments.

Encapsulation efficiency and size distribution of the liposomalall-trans retinoic acid preparation were determined as follows. Theliposomal all-trans retinoic acid preparation was centrifuged at30,000×g for 45 minutes. A yellowish pellet containing the retinoic acidand the lipids was obtained. By light microscopy, the pellet wascomposed of liposomes with no crystals or drug aggregates. Theencapsulation efficiency was calculated to be greater than 90% bymeasuring the amount of free retinoic acid in the supernatant by UV.spectrophotometry. Liposomes were sized in a Coulter-Counter andChannelizer. The size distribution was as follows: 27% of liposomes lessthan 2 micrometers (μm), 65% between 2 μm and 3 μm, 14% between 3 μm and5 μm, 1% more than 5 μm. The method used for encapsulation of retinoidswas simple, reproducible and could be used for large scale production,for example, for clinical trials.

Further experiments were performed by the same procedure but withdifferent lipids, ratios of lipids and the use of ³ H-all-trans retinoicacid. Additional lipids utilized were dipalmitoyl phosphatidyl choline(DPPC) stearylamine (SA) and cholesterol. After sedimentation of theliposomes, residual ³ H was determined and encapsulation efficiencycalculated. Table 1 shows encapsulation efficiencies determined by thismethod for various L-RA preparations.

                  TABLE 1                                                         ______________________________________                                        Encapsulation Efficiency of                                                   Retinoic Acid in Liposomes                                                                      ENCAPSULATION EFFICIENCY                                    LIPOSOME COMPOSITION                                                                            (%)                                                         ______________________________________                                        DMPC:cholesterol 9:1                                                                            69.3                                                        DMPC:cholesterol 9:3                                                                            64.5                                                        DPPC              69.1                                                        DMPC:SA:cholesterol 8:1:1                                                                       56.7                                                        DMPC:DMPG 7:3     90                                                          DMPC:DMPG 9:1     90.7                                                        ______________________________________                                    

Of the lipid compositions studied, DMPC:DMPG at ratios between 7:3 and9:1 gave superior encapsulation efficiencies. Liposomal all-transretinol (L-ROH) was prepared by the methods described above for L-RAwith DMPC:DMPG, 7:3.

EXAMPLE 2 Stability of Liposomal Retinoic Acid

Liposomal ³ H-retinoic acid (L-³ H-RA) was prepared with DMPC:DMPG, 7:3as described in Example 1. Samples of the L-³ H-RA were incubated witheither phosphate-buffered saline (PBS) or PBS with 20% (by volume) fetalcalf serum (FCS). After various periods of incubation at about 37° C.,aliquots were removed and centrifuged to sediment liposomes. The tritiumin the supernatant solution was measured to determine ³ H-RA release.FIG. 1 shows the release of ³ H-RA over a two day period. The L-³ H-RAwas over about 80% stable over the period of the experiment, even in thepresence of 20% FCS.

When ³ H-all-trans retinol was used to label L-ROH and stability in PBSmeasured, only about 5% of the ³ H-ROH was released after a 24 hrincubation at 37° C.

EXAMPLE 3 In Vitro Lysis of Human Erythrocytes (RBCS) by Retinoic Acidor Liposomal Retinoic Acid

Lysis of human red blood cells (RBCs) was quantitated by measuring therelease of hemoglobin in the supernatants by observation of increases inoptical density at 550 nanometers (nm), as described previously (Mehta,et al., Biochem. Biophys, Acta., Vol. 770-, pp 230-234 (1984). Free-RAdissolved in dimethyl formamide (DMFA), was added to the RBCs. Resultswith appropriate solvent controls, empty liposomes, and empty liposomesplus free-drug were also noted. Release of hemoglobin by hypotonic lysisof the same number of human RBCs by water was taken as a 100% positivecontrol, while cells treated with PBS were taken as negative controls.

Preparations of L-RA comprising various lipids were incubated at aconcentration of 20 microgram (μg) RA per ml with RBCs in PBS for 4 hrat 37° C. The toxicity of the L-RA preparations on the basis of percentRBC lysis is shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        In Vitro Toxicity Of L-RA Preparations To RBCs                                LIPOSOME COMPOSITION                                                                             % RBC LYSIS                                                ______________________________________                                        DMPC:Cholesterol   4.5                                                        9:1                                                                           DMPC:Cholesterol   90.2                                                       9:3                                                                           DPPC               6.7                                                        DMPC:SA:Cholesterol                                                                              70.4                                                       8:1:1                                                                         DMPC:DMPG          8                                                          7:3                                                                           DMPC:DMPG          8.3                                                        9:1                                                                           ______________________________________                                    

As may be seen from the data of Table 2, L-RA of DMPC:cholesterol, DPPC,DMPC:DMPG (7:3) and DMPC:DMPG (9:1) exhibited low RBC toxicity underthese conditions. It is of interest to note that the latter two L-RAcompositions exhibited superior encapsulation efficiencies (Table 1).

A further experiment concerning the toxicity over time of free RA andL-RA (DMPC:DMPG-7:3) toward RBC was conducted. Human erythrocytes wereincubated at 37° C. in PBS with 10 ug/ml free RA or 120 ug/ml L-RA, andRBC lysis monitored over a period of 5 hr. FIG. 2 shows time courses ofRBC lysis. At between about 1 hr and about 3 hr, the free RA extensivelylysed a large majority of the erythrocytes. When a similar manipulationwas performed with L-RA (DMPC:DMPG(7:3)) at a RA concentration of 120ug/ml, little RBC lysis occurred (e., less than 10% after 6 hr).

A study was also conducted concerning the effects upon RBC lysis in 2 hrof free RA and L-RA (DMPC:DMPG(7:3)) at various concentrations. FIG. 3shows the results of this study. Free RA showed linearly increasing RBClysis between about 5 ug RA/ml and about 30 ug RA/ml. Liposomal RAcaused RBC lysis of only about 5% at a concentration of 160 ug RA/ml.

EXAMPLE 4 Acute Toxicity of Free and Liposomal Retinoic Acid

The acute toxicity of free and liposomal all-trans retinoic acid wasstudied in CD1 mice. Free all-trans retinoic acid was prepared as anemulsion in normal saline containing 10% DMSO and 2% Tween 80 at aconcentration of 3 to 5 mg/ml. Liposomal all-trans retinoic acid wasprepared using a lipid:drug ratio of 15:1. The final concentration ofall-trans retinoic acid in the liposomal preparation was 3 mg/ml. Emptyliposomes of the same lipid composition (DMPC:DMPG 7:3) were also testedat doses equivalent to 80 mg/kg, 100 mg/kg, and 120 mg/kg ofliposomal-all trans retinoic acid. Normal saline containing 10% DMSO and2% Tween 80 was also tested as a control at a dose equivalent to 50mg/kg of free all-trans retinoic acid. All drugs tested were injectedintravenously via tail vein as a single bolus. The injected volumes offree and liposomal-all-trans retinoic acid were the same for each dose.

Table 3 shows data obtained from these acute toxicity experiments.

                  TABLE 3                                                         ______________________________________                                        Acute Toxicity of Free and                                                    Liposomal All-Trans Retinoic Acid                                                                   Number    Number                                                    Dose      Animals   Animals                                       Drug        (mg/kg)   with seizures                                                                           alive (72 hr)                                 ______________________________________                                        Free RA     10        0/6       6/6                                                       20        6/6       5/6                                                       30        6/6       4/6                                                       40        3/3       0/3                                                       50        3/3       0/3                                           L-RA        40        0/6       6/6                                                       60        0/6       6/6                                                       80        0/6       6/6                                                       100       0/6       6/6                                                       120       0/6       6/6                                           Empty Liposomes                                                                           80        0/6       6/6                                                       100       0/6       5/6                                                       120       0/6       6/6                                           Normal saline                                                                 10% DMSO                                                                      2% Tween 80 50        0/6       6/6                                           ______________________________________                                    

The maximum non-toxic dose of free all-trans retinoic acid was 10 mg/kg.Higher doses caused seizures immediately after injection. The acute LD₅₀(deaths occurring up to 72 hours after injection) of free all-transretinoic acid was 32 mg/kg. The cause of death was cardiopulmonaryarrest after seizures for 1-2 minutes in all animals. No seizures ordeaths were observed in the animals treated with liposomal all-transretinoic acid at a dose of 120 mg/kg (maximum non-toxic dose and LD₅₀greater than 120 mg/kg). Higher doses were not tested. No seizures wereobserved in the animals treated with empty liposomes or normal salinewith 10% DMSO and 2% Tween 80.

EXAMPLE 5 In Vitro Inhibition of Tumor Cell Growth

Liposomal all-trans retinoic acid (L-RA) was prepared as described inExample 1.

Cells of the human monocytic cell line THP-1 were inoculated intosamples of eucaryotic cell culture medium in the presence or absence ofL-RA, at a final RA concentration of 1 micromolar (μm). After 24 hr at37° C., ³ H-thymidine was added to each culture and incorporationthereof into cellular polynucleotides measured. Table 4 shows thepercentage of tumor growth inhibition as reflected by decreases in ³H-thymidine incorporation induced by L-RA of differing lipidcompositions.

                  TABLE 4                                                         ______________________________________                                        L-RA Inhibition of Tumor Cell Growth                                                           TUMOR CELL (THP-1)                                           LIPOSOME COMPOSITION                                                                           INHIBITION (%)                                               ______________________________________                                        DMPC:Cholesterol 72                                                           9:1                                                                           DMPC:Cholesterol 22                                                           9:3                                                                           DPPC             8                                                            DMPC:SA:Cholesterol                                                                            84                                                           8:1:1                                                                         DMPC:DMPG        70                                                           7:3                                                                           DMPC:DMPG        32                                                           9:1                                                                           ______________________________________                                    

From Table 4, it should be noted that L-RA (DMPC:DMPG-7:3), which, aspreviously shown herein, gave a superior encapsulation efficiency andshowed-a low RBC toxicity (Tables 1 and 2), also effectively inhibitedthe tumor cell growth.

Cells of the human monocytic cell line THP-1 and of the humanhistiocytic cell line U-937 were inoculated at about 20,000 cells percell in aliquots of eucaryotic cell culture medium contained in wells ofa 96 well microtiter plate. The medium in various wells containeddifferent amount of free RA or L-RA (DMPC:DMPG 7:3). The cells wereincubated for 72 hr at 37° C. and cell growth determined and compared tothat of controls without any form of retinoic acid. FIG. 4 shows theinhibition of THP-1 cell growth by increasing concentrations of free RAor L-RA (DMPC:DMPG 7:3). At concentrations of less than 1 μg RA/ml, bothpreparations inhibited cell growth by over 90%.

The human monocytic leukemia THP-1 cells, after a 72 hr incubation witheither free RA or L-RA at a concentration of 0.3 μg RA/ml, were observedto have lost their generally ovate form and to have a more flattened andspread morphological appearance often associated with cellulardifferentiation. The generally ovate form was retained when the cellswere cultured in the absence of any free or liposomal retinoic acid.

After incubation for 24 hr with 0.3 μg/ml or 0.6 μg/ml RA or L-RA inanother experiment, THP-1 cells had increased levels of tissuetransglutaminase enzymic activity, a marker for monocytic celldifferentiation. As shown in FIG. 5, THP-1 cells, at 4×10⁶ cells/ml,showed about 500% greater transglutaminase activity when incubated withL-RA as compared to free RA at equivalent retinoic acid concentrations.

Cells of the human histiocytic cell line U-937 were distributed andcultured under the same conditions as the THP-1 cells in the priorexperiment. FIG. 6 shows the effects upon cell growth of increasingconcentrations of free all-trans retinoic acid (RA), liposomal(DMPC:DMPG 7:3) all-trans retinoic acid (L-RA) and empty liposomes(which were devoid of retinoic acid). It should be noted that the U-937cells were almost completely growth-inhibited by L-RA at a retinoic acidconcentration of about 10 ug/ml while this amount of free RA inhibitedgrowth less than 50%.

EXAMPLE 6 Antitumor Activity of Liposomal All-Trans Retinoic Acid invivo

The antitumor activity of liposomal-all trans retinoic acid (DMPC:DMPG7:3) was tested in vivo against liver metastases of M5076reticulosarcoma. C57BL/6 mice were inoculated with 20,000 M5076 cells onday 0. Intravenous treatment with 60 mg/kg liposomal all-trans retinoicacid was given on day 4. The mean survival of control animals(non-treated) was 21.8+1.6 days. The mean survival of treated animalswas 27.0±1.6 days. Liposomal all-trans retinoic acid was shown,therefore, to have antitumor activity at a dose well below the maximumnon-toxic dose, against a cell line (M5076) which was resistant to freeretinoic acid in in vitro studies. THP-1 cells treated in vitro with RA(1 MM) for 72 hours when injected subcutaneously into male mice, failedto develop into tumors, whereas untreated cells formed a huge mass oftumors in such mice.

EXAMPLE 7 Induction of Tissue Transglutaminase in Human Peripheral BloodMonocytes by Intracellular Delivery of Retinoids

Circulating blood monocytes are the precursors of macrophages whichaccumulate at the sites of tumor rejection 2!, delayed hypersensitivity25!, chronic inflammation 6!, and at the site of damaged tissue as apart of the healing processes 11! (see reference citations in sectionD). At these sites, peripheral blood monocytes acquire new functionaland biochemical characteristics that are associated with the maturationor differentiation process. To understand clearly the mechanismsinvolved in differentiation, it is necessary to manipulate theextracellular environment and assess precisely a variety of cellularfunctions and biochemical activities.

Vitamin A and its analogues (retinoids) have been shown to exert aprofound effect on the differentiation of monocytic cells. Both normal19! and leukemic 7,17,28! monocytic cells differentiate in response toretinoids which might suggest that retinoids play a role in regulatingthe differentiation of these cells. According to recent reports, thecellular activity of transglutaminase (TGase), an enzyme that catalyzesthe covalent cross-linking of proteins, may be directly linked to theretinoid's action 4,15,21,23,35,39,39!. Recently, the present inventorsfound that in vitro maturation of human peripheral blood monocytes(HPBM) to macrophage-like cells was associated with the induction andaccumulation of a specific intracellular TGase, tissue TGase 19,22!.Gamma (g)-interferon, which promotes the tumoricidal properties in HPBM,also augmented the expression of tissue TGase 19!. Similarly, theactivation of guinea pig and mouse macrophages in vivo was associatedwith a marked increase in tissue TGase activity 10,24,34!. Terminaldifferentiation of human monocytic leukemia cells (THP-1) induced byphorbol ester and retinoic acid was associated with induction andaccumulation of tissue TGase (17!, suggesting that the induction oftissue TGase was a marker of monocytic cell differentiation. The presentinvention involves further definition of the role of retinoids indifferentiation and maturation of HPBM and comprises studies of cultureconditions that inhibit or facilitate the internalization of retinoidsby HPBM on expression of tissue TGase. The studies herein demonstratethat HPBM, isolated into two subpopulations, show no significantdifference in their ability to express tissue TGase activity induced byeither in vitro culture or exposure to recombinant interferon gamma(rIFN-g), and that the expression of tissue TGase in cultured HPBM maybe induced by a direct delivery of retinoids to intracellular sites.

A. Materials and Methods

1. Materials

RPMI-1640 medium supplemented with L-glutamine and human AB serum werefrom Gibco Laboratories (Grand Island, N.Y.); Escherichia coli-derivedhuman recombinant g-interferon (rIFN-g) was kindly supplied by GenentechInc. (South San Francisco, Calif.); and all-trans retinol (ROH) andall-trans retinoic acid (RA) were purchased from Sigma Chemical Co. (St.Louis, Mo.). The chromatographically pure lipids dimyristoylphosphatidyl choline (DMPC) and dimyristoyl phosphatidyl glycerol (DMPG)were from Avanti Polar Lipids (Birmingham, Ala.); tritiated putrescine(sp. act. 28.8 Ci/mmol), from New England Nuclear (Boston, Mass.); andtritiated ROH (sp. act. 15 mci/mmol), from Amersham (Arlington Heights,Ill.). Lipids, culture medium, and serum were screened for endotoxinwith the Limulus amebocyte lysate assay (MA Bioproducts, Walkersville,Md.), and they were used only when endotoxin contamination was less than0.25 ng/ml.

2. HPBM Isolation, Purification, and Culture

Pure populations of HPBM were obtained by countercurrent centrifugalelutriation of mononuclear leukocyte-rich fractions obtained from normaldonors who were undergoing routine plateletpheresis 12!. HPBM wereisolated into two subpopulations according to size with a Coulter ZBIcounter and C-1000 channelizer (Coulter Electronics, Hialeah, Fla.). Themedian volume of small monocytes was 255 mm³, and that of the largemonocytes was 280 mm³. The small monocytes were 95%±3% nonspecificesterase-positive and the large monocytes were 98%±2% positive. Detailedprocedures for isolation and characteristics of these subpopulationshave been published elsewhere 36,37!. Small, large, or mixed (obtainedby mixing equal parts of small and large HPBM) HPBM subpopulations werewashed once with medium (RPMI-1640 supplemented with L-glutamine, 20 mMHEPES buffer, 20 ug/ml gentamicin, and 5% human AB Serum) andresuspended to 0.5 million/ml density in the same medium. The cells weredispensed in 4-ml samples into 35-mm-well plates and cultured underappropriate conditions.

3. Enzyme Assay

Tissue TGase activity in cell extracts was measured as a Ca²⁺, dependentincorporation of ³ H! putrescine into dimethylcasein. In brief, culturedHPBM were washed three times in Tris-buffered saline (20 mM Tris-HCl,0.15M NaCl, pH 7.6) and scraped from the dish in a minimal volume of thesame buffer containing 1 mM EDTA and 15 mM Beta-mercaptoethanol. Thecells were lysed by sonication, and TGase activity in the lysates wasdetermined as described previously 13,20!. The protein content in celllysates was determined by Lowry's method 14! with bovine gamma globulinas standard. The enzyme activity was expressed as nanomoles ofputrescine incorporated into dimethyl-casein per hour per milligram ofcell protein.

4. Immunochemical Detection of Tissue TGase

To detect tissue TGase in cell extracts, the cell lysates weresolubilized in 20 mm Tris-HCl (pH 6.8) containing 1% sodium dodecylsulfate (SDS), 0.75M Beta-mercaptoethanol, 2.5% sucrose and 0.001%bromophenol blue. Solubilized extracts were fractionated byelectrophoresis on a 6.5% discontinuous polyacrylamide gel andelectroblotted onto nitrocellulose paper. The paper was neutralized with5% bovine serum albumin and treated with iodinated anti-tissue TGaseantibody; the preparation, characterization and properties of thisantibody have been described elsewhere (24!. The unbound antibody wasremoved by washing the paper in Tris-HCl buffer (50 mM, pH 7.5)containing 200 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.1% SDS, and0.25% gelatin, and the paper was dried and autoradiographed as describedearlier 20,24!.

5. Preparation of Liposomes

Multilamellar vesicles (liposomes) containing DMPC and DMPG at a molarratio of 7:3 were prepared as described 16,18!. All-trans ROH or RA wereencapsulated by adding the required amount of the drug (predissolved inethanol) in lipid-containing organic solvents before vacuum drying. Thedried lipid-drug film was dispersed by agitation in sterile salinesolution. Retinoids up to a 1:10 drug:lipid ratio could be completelyencapsulated within the liposomes and were highly stable. The stabilityand encapsulation efficiency of the liposome preparations were studiedby using radiolabeled retinol and showed that only 5%±2% of theincorporated radioactivity leaked out in the supernatant after 24-hrincubation at 37° C.

6. Binding Assay for ³ H!ROH

Freshly isolated HPBM were cultured in serum containing medium alone ormedium plus 50 units (U)/ml rIFN-g for varying periods of time. At theend of indicated time periods, HPBM monolayers were washed twice in icecold medium and resuspended in 0.5 ml of prechilled reaction mixturecontaining 5.0 microcuries (μCi)/ml 11,12(n)³ H! vitamin A (free ROH) inRPMI medium supplemented with 5% delipidized human AB serum (serumdelipidization was done by organic solvent extraction as describedearlier 33!. Binding assays were carried out for 1 hr in an ice bath.After a 1-hr incubation, the monocyte monolayers were washed six timeswith ice-cold medium and the cells were lysed in 200 μl of Triton X-100.Fifty-microliter aliquots of cell lysates, in triplicate, were countedfor the cell-associated radioactivity. Background counts, obtained byadding the reaction mixture toward the end of the 1-hr incubation beforeharvesting, were subtracted from the experimental values.

B. Results

1. Tissue TGase Induction During In Vitro Culture of HPBM

The culture of HPBM in the presence of serum-containing medium for up to10 days was associated with a marked induction of tissue TGase activityin both small and large HPBM (FIG. 7), the increase in enzyme activitybeing more rapid after about 4 days of culture. After 10 days inculture, small monocytes showed a 93-fold increase in enzyme activity(from 0.44 to 41.1 nmol/hr/mg), whereas large HPBM accumulated about103-fold increase in the enzyme activity (from 0.36 to 37.4 nmol/hr/mg).Small and large HPBM mixed together and cultured under similarconditions showed no significant difference in the rate and amount ofaccumulation of tissue TGase activity compared with that of individualHPBM fractions (data not shown). Induction of enzyme activity wasassociated with a change in the morphology of cultured monocytes.Freshly isolated HPBM looked rounded, but after 6-8 days in culture boththe large and small HPBM became firmly adherent to the plastic surface,were more spread and flattened, and had the appearance typical of maturemacrophages. By day 10, when the cells had accumulated maximal levels ofenzyme activity, these levels then either plateaued or starteddeclining.

2. Effect of rIFN-g on Tissue TGase Expression

The effect of continuous exposure to rIFN-g on induction of tissue TGaseactivity in HPBM is shown in FIG. 8. Small and large monocytes werecultured in serum-containing medium for 72 hr in the presence ofincreasing concentrations of rIFN-g. Enzyme activity in the HPBMpopulations increased significantly after their continuous exposure torIFN-g compared with that of cells cultured in the presence of mediumalong. However, rIFN-g dose size produced no significant difference inenzyme activity between the two HPBM populations. As previously noted19!, a 100-U/ml dose of rIFN-g seemed to be optimal for augmenting TGaseactivity; higher rIFN-g-concentrations were less effective. Theinductive effect of rIFN-g on tissue TGase activity was evidence at 5U/ml and pretreatment of HPBM cultures with rIFN-g (100 U/ml) followedby washing and subsequent culture in medium alone did not enhance theexpression of tissue TGase. The rIFN-g-induced augmentation of tissueTGase was associated with morphologic changes in HPBM so that therIFN-g-treated cells were more spread out and flattened than theuntreated control cells after three days in culture.

3. Effect of Retinoids on Tissue TGase Induction

Since the two HPBM populations showed no heterogeneity in terms ofinduced tissue TGase levels, our subsequent studies were done with wholeHPBM fraction without separation into subsets. HPBM cultured in thepresence of 500 nM RA for 24 hr accumulated at least three-fold higherenzyme activity than did the control cells cultured in medium along(FIG. 9). Continuous exposure to RA caused a rapid and linear increasein the enzyme activity, whereas in the control cells no significantchange in the level of tissue TGase activity was observed for up to 2days of culture. By day 3, the control cells accumulated about six-foldhigher enzyme activity (3.4 nmol/hr/mg) than did freshly isolated HPBM(0.6 nmol/hr/mg), but they still had significantly less enzyme activitythan the RA-treated cells (9.8 nmol/hr/mg). Retinoic acid-inducedexpression of tissue TGase was dose dependent (FIG. 9 inset). ROH, thephysiologic analogue of RA, did not induce the expression of tissueTGase in HPBM even at a dose of 1 μM. Thus, HPBM cultured in thepresence of ROH for up to 3 days showed no significant difference inaccumulation of tissue TGase activity when compared with that of controlcells cultured in medium along (FIG. 9).

4. Effect of Liposome-Encapsulated Retinoids on Tissue TGase Induction

Liposome-encapsulated RA was more effective in inducing tissue TGaseexpression than was free RA at an equimolar concentration. After 24-hrculture, the amount of tissue TGase activity in HPBM induced by free orliposomal RA at an equimolar concentration of 500 nM was notsignificantly different (3.4 and 3.7 nmol/hr/mg, respectively); after 48and 72 hr, however, liposomal RA-treated cells accumulated at least 50%more enzyme activity than did free RA-treated cells (FIG. 10A). Thatincrease in enzyme activity by liposome-encapsulated RA was a specificeffect of RA and not of lipids was demonstrated by the fact that aculture of HPBM in the presence of "empty liposomes," and containingequivalent amount of lipids did not induce enzyme activity throughoutthe incubation period. "Empty liposomes," as reported earlier 20!,inhibited serum-induced expression of tissue TGase after 72 hr ofculture (FIG. 10A). The free or liposomal RA-induced increase in enzymeactivity was caused by an increased amount of the enzyme peptide, asrevealed by Western-blot analysis of cell lysates using a iodinatedantibody to tissue TGase (FIG. 10B). The increase in enzyme activity wasproportional to the increase in enzyme peptide and not caused byactivation of preexisting enzyme.

Retinol, which in its free form was unable to enhance the expression oftissue TGase in HPBM, became active when presented in liposomal form.Liposome-encapsulated ROH caused a rapid and linear increase in tissueTGase activity with time in culture (FIG. 11A). After 72 hr of culture,liposomal-ROH caused a nine-fold increase in enzyme activity (7.1nmol/hr/mg) when compared to that of control cells exposed to free ROHunder similar conditions (0.8 nmol/hr/mg). Liposomal ROH-inducedexpression of tissue TGase resulted from increased accumulation of theenzyme peptide as demonstrated by Western-blot analysis (FIG. 11B).

5. Tissue TGase induction is Related to HPBM Uptake of Retinoids

The effect of in vitro maturation and rIFN-g treatment on the binding oftritiated-ROH by HPBM was examined. After 4 days of control culture(medium dose), tritiated-ROH binding by HPBM increased 50% compared tothis binding by freshly isolated cells. After 9 days the control culturebinding value increased to 350%. The increases in ROH binding wereassociated with parallel increases in tissue TGase activity (Table 5).

                  TABLE 5                                                         ______________________________________                                        Effect of In Vitro Culture and rIFN-g                                         Treatment on  .sup.3 H! ROH Binding by HPBM                                                          .sup.3 H! ROH bound                                                                      Tissue TGase                                           Days in    (cpm/10 μg                                                                             activity                                    Culture Conditions                                                                       Culture    protein)    (nmol/hr/mg)                                ______________________________________                                        medium alone                                                                             0          684 ± 25 0.25 ± 0.13                                         4           994 ± 115                                                                             2.96 ± 0.75                                         9          2,220 ± 144                                                                            32.60 ± 8.50                             medium alone                                                                             3          626 ± 37  2.9 ± 0.23                              medium + rIFN-g                                                                          3          1,782 ± 130                                                                            7.6 ± 0.7                                ______________________________________                                         .sup.a HPBM were cultured in serumcontaining medium alone or medium           containing 50 U/ml rIFNg for indicated periods of time.                       .sup.b Binding of tritiated ROH during different periods of culture was       determined as described in Materials and Methods.                             .sup.c Parallel cultures of HPBM maintained under similar conditions were     used for assaying enzyme activity as described in Materials and Methods. 

Exposure of HPBM to rIFN-g augmented the ROH binding and the expressionof enzyme activity. The rIFN-g-treated cells showed a threefold higher ³H!ROH binding than did control cells incubated in the presence ofserum-containing medium alone for the same period of time. The presenceof delipidized serum in the reaction mixture was essential; only 10% ofthe total counts were cell-associated when delipidized serum was omittedfrom the reaction mixture.

C. Discussion

The results reported in this Example suggested that HPBM, isolated intotwo populations based on their size and density, have equal potential todifferentiate into mature macrophages. The in vitro maturation of HPBMto macrophages was associated with enhanced binding and uptake ofretinol, presumably as a result of the acquisition of cell surfacereceptors for serum retinol-binding protein. Exposure of HPBM to rIFN-gfor 72 hr led to enhanced binding of ³ H!ROH that was comparable to thebinding activity of control HPBM cultured in vitro for 9 days. HPBMmaturation induced by in vitro culture or by exposure to rIFN-g wasaccompanied by similar morphologic and enzymatic changes. Therequirement of cell surface receptor for serum retinol-binding proteincould be circumvented by direct intracellular delivery of ROH.

Recently, several reports have suggested an association betweenmonocytic cell differentiation and induction of tissue TGase10,17,19,21-24,34!. Freshly isolated HPBM that have very low levels oftissue TGase accumulate large amounts of this enzyme after their invitro maturation 19,22!. Just as the two subpopulations of HPBM showedno significant difference in their ability to induce and accumulatetissue TGase activity during in vitro differentiation to macrophages,both fractions were equally responsive to the effect of rIFN-g in termsof augmented enzyme expression (FIG. 8). Functional heterogeneity amongHPBM subpopulations isolated by similar criteria has been reportedearlier. Thus, the subsets of HPBM isolated into small and largepopulations have been reported to produce different amounts of reactiveoxygen species 37!, prostaglandins 1,30!, antibody dependentcell-medicated cytotoxicity 27!, and tumor-cell killing 26!. Thisfunctional heterogeneity among HPBM subpopulations has been attributedto either maturational or clonal differences. The data presented herein,however, suggest no heterogeneity among HPBM subpopulations in inductionof tissue TGase, a marker for monocytic cell differentiation, and equalpotential for differentiating into mature macrophages. The ability ofrIFN-g to enhance tissue TGase expression in both HPBM subpopulationssuggests that this endogenous cytokine may play an important role in thematuration, differentiation, and expression of differentiated functionsin monocytic cells.

The factors in serum responsible for induction and accumulation oftissue TGase in cultured HPBM and macrophages have been shown to beendogenous retinoids and serum retinol-binding protein 21!. Extractionof retinoids by delipidization or depletion of retinol-binding proteinfrom the serum completely abolished its enzyme-inducing ability 19,21!.Serum retinol-binding protein is believed to be responsible forintravascular transport and delivery of retinol to specific targettissues 8,9,29,31!. Receptors for serum retinol-binding protein presenton the surface of target cells are responsible for the specificity ofthe delivery process 9,31!. The binding of ROH-retinol-binding proteincomplex to cell surface receptors apparently facilitates the delivery ofROH into the interior of the cell 9,31!. At superphysiologic doses(greater than 10 nM) on the other hand, RA can enter the cells directlyby simple diffusion without the participation of surface receptors forretinol-binding protein 21!. This suggested that freshly isolated HPBMprobably lack the cell surface receptors for serum retinol-bindingprotein and therefore cannot internalize the endogenous or exogenousretinoids. Indeed, the addition of exogenous RA to HPBM cultures atdoses (e.g. greater than 10 nM) at which the receptor-mediated deliverybecomes irrelevant resulted in a marked induction of tissue TGaseactivity (FIG. 9). The enzyme-inducing ability of RA was augmentedfurther by encapsulating RA within the liposomes and allowing itsinternalization via phagocytosis (FIG. 10).

Of particular interest was the effect of ROH, which, in its free formdid not induce the expression of tissue TGase in freshly isolated HPBM.When ROH was encapsulated within liposomes, however, the requirement fora cell surface receptor for serum retinol-binding protein was obviated.Thus liposomal ROH induced a significant level of tissue TGase activityin HPBM (FIG. 11). This suggested an effective approach for targetingretinol or its inactive analogues to the monocytic cells with no orminimal toxic effects. Because HPBM lack cell surface receptors forserum retinol-binding protein makes administered ROH subject tononspecific internalization by other cell types. The present studiessuggested, furthermore, that interaction of ROH-retinol binding-proteincomplex with the cell surface receptor is required only for theintracellular delivery of retinol and that, unlike in the case of otherhormones 3!, ligand-receptor interaction may not require a secondmessenger for expression of the final event. The increase in TGaseenzyme activity induced by free RA or liposome-encapsulated RA or ROH,was the result of the accumulation of enzyme protein rather than theactivation of preexisting enzyme, as revealed by immunoblots of the celllysates using an iodinated antibody to tissue TGase (FIGS. 10,11).

Preliminary data on tritiated ROH-binding (Table 5) further supportedthe concept that in vitro differentiation of HPBM to mature macrophageswas associated with acquisition of cell surface receptors forretinol-binding protein and that treatment with rIFN-g augmented theexpression of these receptors. Once the HPBM acquire these receptors,they could internalize the endogenous retinoids and induce theexpression of tissue TGase. Indeed, retinoids have been shownspecifically to trigger the gene for tissue TGase in myelocytic cells23!.

Impairment of macrophage function in retinoid-deficient animals has beenwell documented to lead to increased incidence of infections anddecreased tumor-cell killing 5!. In cultures of guinea pig peritonealmacrophages, RA has been reported to increase the intracellular levelsfor the tumoricidal enzyme arginase 32!. The present findings thatretinoids play an important role in the differentiation process of HPBMsupport the idea that retinoids are the important regulators ofmonocyte/macrophage functions.

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28. Olsson, I. L., and Brietman, T. R. Induction of differentiation ofthe human histiocytic lymphoma cell line U-937 by retinoic acid andcyclic adenosine 3', 5'-monophosphate inducing agents. Cancer Res. 42,3924, 1982.

29. Peterson, P. A. Characteristics of a vitamin A transporting proteincomplex occurring in human serum. J. Biol. Chem. 246, 34, 1971.

30. Picker, L. J., Raff, H. V., Goldyne, M. E., and Stobo, J. B.Metabolic Heterogeneity among human monocytes and its modulation ofPGE₂. J. Immunol. 124, 2557, 1980.

31. Rask, L., and Peterson, P. A. In vitro uptake of vitamin A from theretinol-binding plasma protein to mucosal epithelial cells from themonkey's small intestine. J. Biol. Chem. 251, 6360, 1976.

32. Roberts, A. B., and Sporn, M. B. Cellular biology and biochemistryof the retinoids, In The Retinoids, Vol. 2 (Sporn, M. B., Roberts A. B.,and Goodman, D. S., Eds.) New York: Academic Press, p. 209, 1984.

33. Rothblat, G. H., Arborgast, L. Y., Quellett, L., and Howard, B. V.Preparation of delipidized serum proteins for use in cell culturesystems. In Vitro 12, 554, 1976.

34. Schroff, G., Neumann, C., and Sorg, C. Transglutaminase as a markerfor subsets of murine macrophages. Eur. J. Immuno. 11, 637, 1981.

35. Scott, K. F., Meyskens, F. L., Jr., and Russel, D. H. Retinoidsincrease transglutaminase activity and inhibit ornithine decarboxylaseactivity in Chinese ovary hamster cells and in melanoma cells stimulateto differentiate. Proc. Natl. Acad. Sci. USA, 79, 4053, 1982.

36. Turpin, J., Hester, J. P., Hersh, E. M., and Lopez-Berestein. G.Centrifugal elutriation as a method for isolation of large number offunctionally intact human peripheral blood monocytes. J. Clin.Apheresis. 3, 111, 1986.

37. Turpin, J., Hersh, E. M., and Lopez-Berestein, G. Characterizationof small and large human peripheral blood monocytes; effects of in vitromaturation on hydrogen peroxide release and on the response tomacrophage activators. J. Immunol. 136, 4194, 1986.

38. Yuspa, S. Ben, T., and Litchi, U. Regulation of epidermaltransglutaminase activity and terminal differentiation by retinoids andphorbol esters. Cancer Res. 43, 5707, 1983.

39. Yuspa, S., Ben, T., and Steinert, P. Retinoic acid inducestransglutaminase activity but inhibits cornification of culturedepidermal cells. J. Biol. Chem. 257, 9906, 1982.

EXAMPLE 8 In vivo Administration

A. Materials and Methods

1. Liposomes and liposomal all-trans retinoic acid Liposomal all-transretinoic acid was prepared from lyophilized powder in bottles containing3 mg of all-trans retinoic acid and 45 mg of a mixture of twophospholipids, dimyristoyl lecithin and dimyristoyl phosphatidylglycerolin a 3:7 ratio (Avanti Polar Lipids, Birmingham, Ala.). Immediatelybefore use, liposomal all-trans retinoic acid was reconstituted byadding 3 ml of normal saline to each bottle and agitating the suspensionon a vortex mixer for 2-3 min. The reconstituted preparation consistedof multilamellar liposomes (average size, 3.1 μm).

2. Animals

Six-week-old Lewis rats (Charles River. Wilmington, Mass.) were used forthese studies. Groups of eight female rats each were administered 5mg/kg body weight of either all-trans retinoic acid (mixed with mineraloil) orally or liposomal all-trans retinoic acid, intravenously (in tailvein). Each rat received a total of 15 doses, twice a week, 3-4 daysapart, for 7 weeks. Blood samples of 150 μl were collected from the tailvein 5, 30, 60, and 90 minutes following administration of the last doseand analyzed for all-trans retinoic acid levels by HPLC. Blood sampleswere also collected 60 min after administration of the first and sixthdose and analyzed for all-trans retinoic acid. Ninety minutes after thelast dose, all the animals were killed and blood samples of 3 ml werecollected to study the hematologic and blood chemistry parameters.Sections of tissues were collected on dry ice for further processing orwere fixed in formalin for histopathologic analysis.

3. Cellular retinoic acid-binding protein (CRABP)

Liver samples were collected 90 minutes after the last dose andprocessed individually. Total CRABP (CRABP I and II) levels werequantitated by slab gel electrophoresis. Briefly, cytoplasmic proteinswere extracted and 100-200 μg protein were incubated overnight at 4° C.in a 100 μl solution of 50 nM ³ H!-all-trans retinoic acid (specificactivity 49.3 Ci/mmol; and 2 mM dithiothreitol with or without 200-foldexcess of unlabeled all-trans retinoic acid. Reactants were fractionatedover vertical slab gel polyacrylamide electrophoresis under nativeconditions. After electrophoresis the gel was divided into lanes and cutinto 5 mm bands; radioactivity was assessed in a liquid scintillationcounter. Specific binding was determined from the radioactivityrecovered with or without the 200-fold excess of unlabeled retinoid.

4. In vitro metabolism of all-trans retinoic acid

Liver samples obtained from the animals at the time of death were rinsedin ice-cold saline and homogenized individually in a 3-fold volume of0.25M sucrose 0.05M Tris-HCl (pH 7.4) using a Teflon® glass homogenizer.Microsomes were isolated by differential centrifugation at (10,000 g for20 min; 100,000 g, 60 min). The microsomal pellet was suspended in 0.05MTris-HCl (pH 7.4), portioned into aliquots and stored at -70° C. Proteincontent was determined by Biorad Protein Assay using bovine serumalbumin as the standard.

The assay buffer and conditions used for determining the ability ofmicrosomes to metabolize carboxyl-¹⁴ C! all-trans retinoic acid(specific activity 13.7 Ci/nmol) were essentially the same as thosedescribed by Van Wauwe et al., J. Pharmac. exp. Ther., Vol. 245, 718.After 30 min, the reaction was stopped by cooling and the samples werelyophilized to dryness. Dried residues were extracted with methanolcontaining butylated hydroxyanisole (0.05%, v/v), and the extracts wereevaporated and redissolved in small volumes of methanol (25-50 μl).All-trans retinoic acid and metabolites were then separated by thinlayer chromatography by spotting 20,000-25,000 cpm on 0.25-mmsilica-coated plastic sheets, and developing in a solution of benzene,chloroform, and water (4:1:1). The radioactive spots were located byspraying, the plates with EN³ Hance (New England Nuclear) andautoradiography. The radioactive bands were scraped out, extracted withSolvable (New England Nuclear) and counted in a scintillation counter.The extent of all-trans retinoic acid metabolism was determined from theproportions of cpm in appropriate zones and expressed as a percentage ofthe total amount of radioactivity recovered.

5. HPLC analysis

The extent of all-trans retinoic acid metabolism by isolated livermicrosomes was also determined by HPLC analysis. The reactants werelyophilized and the residues were extracted twice with 2 ml of methanolcontaining 0.05% butylated hydroxyanisole (Sigma Chemical Co., St.Louis, Mo.). After centrifugation, the supernatants were aspirated andevaporated. The resulting pellets were re-extracted in amethanol:dichloromethane solution (75:25) and again evaporated in vacuo.More than 80% of the added all-trans retinoic acid was recovered. Thefinal pellet was mixed with 200 μl of mobile phase for reverse-phaseHPLC. A portion of each sample (150 μl) was analyzed on a 10-μm C₁₈Bondapack column (3-9×300 mm; Waters Associates, Farmingham, Mass.).Samples were eluted with a solution of methanol, water, and formic acid(60:40:0.05) containing 10 mM ammonium acetate at a flow rate of 2ml/min. After 20 min, the solvent was changed to 100% methanol in orderto elute all-trans retinoic acid.

Reverse phase HPLC was also used for determining the bloodconcentrations of all-trans retinoic acid. All procedures were performedin a room with the lights dimmed. Whole blood samples (200 μl) wereextracted twice (1 ml each) with methanol. After centrifugation, thesupernatants were vacuum-dried and the dried pellets were reconstitutedin 200 μl of methanol. The recovery of all-trans retinoic acid underthese conditions was calculated to be 85%±7%. The HPLC system includedtwo pumps and a Zorbax-C8 reverse phase column (4 mm×8 cm; Supelco,Pa.). The mobile phase consisted of a linear gradient between solvent A(THF and water (25:75) containing 0.04% ammonium acetate. pH 4) andsolvent B (100% THF) during a 16 minute run at a flow rate of 1.8ml/min. The absorbance was monitored at 346 nm. Retention time forall-trans retinoic acid under these conditions was approximately 9.8minutes.

6. Determination of P450 levels

Cytochrome P450 levels in liver microsomes were determinedspectrophotometrically. The assay system is based on the carbon monoxide(CO) difference spectra of dithionite-reduced samples, assuming a valueof 91 mM/cm for the molar extinction increment between 450 and 490 mμ.The P450 activity was calculated by the following formula: (change inabsorbance between dithionite-reduced sample and CO samplealone)/91×1000: it was expressed as nm/mg protein.

7. Statistical analysis

The mean values for the groups were analyzed by using Student's t-testfor paired samples.

B. Results

Hematologic and blood chemistry analysis of samples drawn 90 min. afteradministration of the last dose of liposomal or non-liposomal all-transretinoic acid, summarized in Table 6, revealed no significant changes,except that the number of circulating segmented neutrophils wassignificantly decreased in animals treated with either drug formulation.This decrease in circulating neutrophils was more pronounced in ratstreated with non-liposomal all-trans retinoic acid than in those treatedwith liposomal all-trans retinoic acid or in untreated controls(ρ<0.05). Similarly, no appreciable change was observed in most of theblood chemistry parameters studied, except both the non-liposomalall-trans retinoic acid and liposomal all-trans retinoic acid-treatedrats showed slight increases in alkaline phosphatase levels (Table 6).

                  TABLE 6                                                         ______________________________________                                        HEMATOLOGICAL AND BLOOD CHEMISTRY PARAMETERS                                  OF RATS FOLLOWING LONG-TERM ADMINISTRATION                                    OF FREE ATRA AND L-ATRA*                                                                       ATRA     L-ATRA                                                               (p.o.)   (i.v.)                                                        Control  (5 mg/kg body weight)                                      ______________________________________                                        Hematological                                                                 parameters                                                                    WBCs (× 10.sup.3 /mm.sup.3)                                                         5.1 ± 1.9                                                                             4.3 ± 1.3                                                                             6.3 ± 1.8                                RBCs (× 10.sup.6 /mm.sup.3)                                                         7.1 ± 0.1                                                                             7.0 ± 0.3                                                                             6.6 ± 0.5                                Hgb (gm/dl) 13.5 ± 0.4                                                                            13.3 ± 0.6                                                                            12.6 ± 1.0                               Plts (× 10.sup.3 /mm.sup.3)                                                         607.0 ± 106                                                                           577.0 ± 252                                                                           644.0 ± 107                              Segs (× 10.sup.3 /mm.sup.3)                                                         59.3 ± 4.9                                                                            28.6 ± 8.6                                                                            46.0 ± 14.3                              Lymph (× 10.sup.3 /mm.sup.3)                                                        40.0 ± 4.3                                                                            69.0 ± 10.0                                                                           54.1 ± 9.0                               Blood chemistry                                                               parameters                                                                    Electrolytes (mEq/L)                                                          K.sup.+     3.8 ± 0.2                                                                             4.1 ± 0.2                                                                             3.8 ± 0.4                                Na.sup.+    142.6 ± 0.6                                                                           142.7 ± 0.9                                                                           142.5 ± 1.3                              C1.sup.-    99.3 ± 1.5                                                                            99.0 ± 2.5                                                                            98.7 ± 3.5                               Creatinine (mg %)                                                                         0.43 ± 0.05                                                                           0.56 ± 0.1                                                                            0.51 ± 0.1                               BUN (mg/dl) 21.7 ± 3.2                                                                            22.7 ± 3.1                                                                            22.4 ± 4.7                               SGOT (IU)   171.0 ± 33.1                                                                          144.4 ± 55.2                                                                          131.9 ± 72.1                             SGPT (IU)   62.3 ± 7.6                                                                            76.5 ± 34.0                                                                           73.9 ± 17.9                              Alk. Phos. (IU)                                                                           144.7 ± 5.0                                                                           188.6 ± 23.3                                                                          203.0 ± 20.7                             Bilirubin (mg %)                                                                          0.2 ± 00                                                                              0.16 ± 0.05                                                                           0.13 ± 0.05                              ______________________________________                                         *Groups of rats were administered p.o. free ATRA or i.v LATRA twice a wee     for 7 weeks (15 doses). After the last dose, blood was collected and          analyzed for various parameters. The results shown are the average values     from four to eight rats ± standard deviation from the mean.                WBCs, white blood cells; RBCs, red blood cells; Hgb, hemoglobin; Plts,        platelets; Segs, segmented neutrophils; Lymph, lymphocytes; BUN, blood        urea nitrogen; SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum     glutamic pyruvic transaminase; Alk. Phos., alkaline phosphatase.         

Microscopic examination of tissue sections from the liver, lung, spleen,brain, ovary, skin, kidney, and bone marrow of the treated rats revealedno significant changes in the histopathologic characteristics.Interestingly, spleens from seven of the eight liposomal all-transretinoic acid-treated subjects showed the presence of numerous smallvacuoles throughout the red pulp area. These structures might represententrapped liposomes that were removed during processing. They were seenthroughout the sinusoids and in phagocytes. No such vacuolization wasobserved in animals that were treated with non-liposomal all-transretinoic acid or in control animals treated with saline alone.

FIG. 12(A) shows the levels of all-trans retinoic acid in the blood 60min after oral administration of non-liposomal all-trans retinoic acidor i.v. administration of liposomal all-trans retinoic acid. In general,these blood levels were higher in rats treated with liposomal all-transretinoic acid than in those treated with non-liposomal all-transretinoic acid. This difference became most striking after 7 weeks ofcontinuous drug treatment. The mean level of all-trans retinoic acid inthe blood of rats treated with non-liposomal all-trans retinoic aciddecreased from 3.01±0.33 μg/ml on day 1 to 1.97±0.17 μg/ml (ρ<0.01)after 7 weeks of treatment, whereas the mean blood all-trans retinoicacid levels of rats treated with liposomal all-trans retinoic acid didnot change significantly. The mean all-trans retinoic acid concentrationon day 1 (4.42±1.2 μg/ml) was similar to that at the end of treatment(4.41±0.2 μg/ml). Also studied was blood clearance of all-trans retinoicacid following administration of the last dose of all-trans retinoicacid. Results shown in FIG. 12(B) demonstrate that all-trans retinoicacid could be detected in the blood by HPLC 30 minutes after ingestionof non-liposomal all-trans retinoic acid. The drug reached its maximumlevel (2.01±0.24 μg/ml) after 60 min and remained constant for at least90 min (1.97±0.17 μg/ml). In contrast, significantly higherconcentrations of all-trans retinoic acid (7.57±1.2 ±μg/ml) wereobserved in the blood 5 min following i.v. administration of liposomalall-trans retinoic acid. The clearance of liposomal all-trans retinoicacid from blood occurred in two phases, the initial rapid phase(t_(1/2)α =16 min) followed by a slower terminal phase (t_(1/2)β =55min). Nonetheless, blood levels of the drug were significantly higher insubjects treated with liposomal all-trans retinoic acid at each timepoint studied (ρ<0.001) than in animals administered non-liposomalall-trans retinoic acid.

Particularly addressing FIG. 12: Blood concentrations of all-transretinoic acid in rats after 7 weeks treatment with non-liposomalall-trans retinoic acid or liposomal all-trans retinoic acid arepresented. FIG. 12(A) presents data from groups of eight ratsadministered (5 mg/kg body weight) either p.o. non-liposomal all-transretinoic acid (cross-hatched bars) or i.v. liposomal all-trans retinoicacid (solid bars) twice a week for a total of 7 weeks. Blood samples(200 μl) were collected 60 min after the administration of the first,sixth, and fifteenth doses, and 150 μl aliquots of the blood wereanalyzed for all-trans retinoic acid by HPLC. In FIG. 12(B) data ispresented following administration of the last dose. Blood samples werecollected from animals treated with non-liposomal all-trans retinoicacid (open circles) or liposomal all-trans retinoic acid (solid dots) atindicated time intervals and analyzed by HPLC for all-trans retinoicacid content. The results shown are mean plasma drug concentrations insix rats±S.D.

Because cytochrome P450-dependent accelerated catabolism and inductionof CRABP have been implicated in the acquisition of clinical resistanceto all-trans retinoic acid, a determination was made that the CRABP andcytochrome P450 levels in liver tissues of rats that had been treatedwith either all-trans retinoic acid formulation was mad. No appreciabledifferences in CRABP levels were observed between liver samples of ratsthat had been treated with non-liposomal all-trans retinoic acid orliposomal all-trans retinoic acid and untreated controls. Similarly,there were no significant changes in total cytochrome P450 levels inliver microsomes from rats treated with non-liposomal all-trans retinoicacid (0.63±0.13 nmol/mg; n=7) or liposomal all-trans retinoic acid(0.59±0.01 nmol/mg; n=7) or untreated rats (0.68±0.15 nmol/mg; n=6).

In vitro, however, the liver microsomes isolated from rats that weretreated with non-liposomal all-trans retinoic acid exhibited significantrapid catabolism of all-trans retinoic acid. Incubation of ¹⁴C!all-trans retinoic acid with isolated liver microsomes in the presenceof NADPH resulted in rapid conversion of all-trans retinoic acid intotwo polar products as determined by thin layer chromatography.Incubation under similar conditions of liver microsomes from untreatedrats or rats treated with liposomal all-trans retinoic acid revealed asignificantly slower rate of metabolism of all-trans retinoic acid intothese polar metabolites. When combined, these metabolites accounted forabout 33±0.8% of the microsomes from untreated rats and 28.8±2.57% ofthose from liposomal all-trans retinoic acid-treated rats, whereas theyaccounted for 57±11.2% of the microsomes from animals treated withnon-liposomal all-trans retinoic acid. (FIG. 13(A)) Individual valuesfor intact all-trans retinoic acid and its polar metabolites generatedin the presence of NADPH by liver microsomes that were isolated fromfive different rats treated with either non-liposomal all-trans retinoicacid or liposomal all-trans retinoic acid or from three untreated ratsare shown in FIG. 13 (B). Microsomes from all liposomal all-transretinoic acid-treated and control animals induced much slower catabolismof all-trans retinoic acid than those from rats administerednon-liposomal all-trans retinoic acid (FIG. 13(B)). Liver microsomesisolated from rats that were treated with "empty liposomes" withoutall-trans retinoic acid showed rates of conversion of all-trans retinoicacid to its metabolites similar to those of the untreated controls. Thereaction products generated by incubating all-trans retinoic acid in thepresence of NADPH and liver microsomes were further analyzed by reversephase HPLC. Results of that HPLC analysis demonstrated that microsomesfrom rats treated with non-liposomal all-trans retinoic acid convertedthe drug into four major products (retention times, 7.5-11.5 min). Twoof the metabolites were eluted at the same positions as authentic 4-ketoall-trans retinoic acid (retention time, 7.8 min) and 4-hydroxyall-trans retinoic acid (retention time, 9.5 min). Incubation ofmicrosomes from rats injected with liposomal all-trans retinoic acidalso converted all-trans retinoic acid into polar metabolites, but thesemetabolites were different, quantitatively and to some extentqualitatively, from those in the group treated with non-liposomalall-trans retinoic acid. For example, the metabolite that eluted at 11.1min from the non-liposomal all-trans retinoic acid microsome reactionmixture was not seen in the liposomal all-trans retinoic acid microsomereaction mixture. Similarly, the amounts of three other products(retention times, 7.8-9.6 min), were much smaller in the reactionmixture incubated with microsomes from liposomal all-trans retinoicacid-treated rats.

Addressing FIG. 13: The effect of long-term all-trans retinoic acidadministration on drug metabolism by liver microsomes is presented. FIG.13(A), at the end of the 7 week treatment period, animals were killedand their liver microsomes isolated. The ability of microsomes tometabolize in vitro ¹⁴ C!all-trans retinoic acid was then determined byincubating microsomes in the presence of NADPH and radiolabeledall-trans retinoic acid (50 nM). The reaction products were fractionatedby thin layer chromatography and extent of drug metabolism wasdetermined by counting the metabolite fractions. Results are expressedas a percentage of all-trans retinoic acid metabolized to polar products(cross-hatched bars) or percentage of all-trans retinoic acid remainingintact (solid bars). The vales shown are averages from five rats±S.D.FIG. 13(B) presents radioactivity (cpm) recovered from intact all-transretinoic acid (lane 1) or its polar metabolites (lane 2), as discussedin FIG. 13(A), were plotted individually for five different rats.

Non-liposomal all-trans retinoic acid has been ineffective inpermanently maintaining the remission state of acute promyelocyticleukemia ("APL"). Even when all-trans retinoic acid administration iscontinued after remission has been achieved, many APL patients stillexperience relapse. Clearly, some mechanism of resistance develops inrelapsed patients whereby the ability of all-trans retinoic acid toinduce cellular differentiation is diminished substantially. Several invitro studies have attempted to explain the evolution of this resistancemechanism, which can be induced in culture after continuous exposure toelevated concentrations of retinoid or carotenoid. Interesting recentclinical pharmacological evidence regarding all-trans retinoic acidresistance (Muindi et al., Blood, 79:299 (1992); Cancer Res., 52:2138(1992)) concluded that the reason for the eventual occurrence of thisretinoid resistance during all-trans retinoic acid therapy isprogressively decreasing plasma drug concentration levels. In mostsubjects the onset of the decrease in plasma drug concentration levelsis within 2-6 weeks after beginning treatment. Although these lowerall-trans retinoic acid plasma levels cannot sustain the differentiationeffects on leukemic cells in vivo, in culture the leukemic cells fromthese patients continue to demonstrate cytodifferentiation sensitivityto all-trans retinoic acid. This resistance is not seen with otherretinoids such as isotretinoin or etretinate.

An advantage of the liposomal all-trans retinoic acid of this inventionis that the lipid formulation bypasses the clearance mechanism thatevolves in the livers of patients treated with the oral formulation.Liposomal formulation is thus not be subject to the same relapse ratesas have been demonstrated in clinical trials of the non-liposomalformulation. In addition, the toxic effects of liposomal all-transretinoic acid should be less severe than those associated withnon-liposomal all-trans retinoic acid because liposome encapsulation ofall-trans retinoic acid decreases direct exposure of the drug duringcirculation to levels below the orally administered toxic dose. Thelatter allows greater total exposure of the drug on initial doseaccompanied by slower clearance of the all-trans retinoic acid from thesite of stem cell seeding.

All-trans retinoic acid is metabolized by a hydroxylation reaction ofthe cyclohexenyl ring, to produce 4-hydroxy metabolites which arefurther oxidized to the 4-oxo metabolites. The hydroxylation ofall-trans retinoic acid to the 4-oxo-all-trans retinoic acid metaboliteis mediated by cytochrome P450-dependent enzymes. The most favoredexplanation of the pharmacological mechanism of all-trans retinoic acidresistance is that continuous all-trans retinoic acid treatment acts toinduce catabolic enzymes that are responsible for conversion of thedrug. Animal studies in which all-trans retinoic acid was administeredin combination with cytochrome P450 enzyme inhibitors (e.g.,ketoconazole or liarozole) showed a significant prolonging of theall-trans retinoic acid plasma half life, thereby supporting thecontention of accelerated enzymatic degradation. The results reportedhere confirm the previous observations that chronic oral administrationof all-trans retinoic acid in rats results in decreased drug plasmaconcentrations, whereas i.v. administration of liposomal all-transretinoic acid at a similar dose and regimen did not alter thepharmacological behavior of the drug and the blood levels remainedstable throughout the study period (FIG. 12). The observed differencesin pharmacological behavior of the two formulations were consistent withinduction of an enzymatic process. Although no differences were observedin total P450 levels in rats treated with either formulation, microsomesfrom rats treated with non-liposomal all-trans retinoic acid metabolizedthe drug much more rapidly than those from rats treated with liposomalall-trans retinoic acid (FIG. 13).

Another factor that might contribute to the retinoid relapse phenomenoninvolves the role of high affinity retinoic acid-binding, proteins CRABPI and II. These proteins are believed to mediate the transfer of theretinoid from cytoplasm to the nucleus of the cell. Increased levels ofCRABP may cause the pooling of retinoids in tissues resulting in lowplasma levels and accelerated clearance of the drug from thecirculation. In normal body tissues the expression of CRABP is thoughtto increase with continuous exposure to retinoids. An increase in CRABPhas been documented in human skin as a result of repeated topicalapplication of all-trans retinoic acid. A similar increase in skin CRABPlevels was also observed by Adamson et al. in rhesus monkeys followingchronic i.v. administration of all-trans retinoic acid.

These authors concluded that the increase in CRABP expression was notrelated to the increase in plasma drug clearance observed withcontinuous all-trans retinoic acid administration, but rather wasrelated to catabolic enzyme induction. In the present data, no increasein levels of liver CRABP was observed in rats administered eithernon-liposomal all-trans retinoic acid or liposomal all-trans retinoicacid on a continuous basis.

The results of Example 8 study, coupled with the following data obtainedin clinical trials discloses that long term oral administration ofall-trans retinoic acid is associated with the rapid clearance of thedrug from plasma that, in turn, contributes to the relapse of thedisease in APL patients, strongly supports the rationale of usingliposomal all-trans retinoic acid to induce long-term remissions in APLpatients.

EXAMPLE 9 In vivo i.v. liposomal all-trans retinoic acid Subjects withhematological malignancies

Liposomal all-trans retinoic acid was administered i.v. over one-halfhour every other day for 28 days to human subjects with hematologicalmalignancies, including T cell cutaneous lymphoma and APL. Dosesinvestigated were 15 mg/M² (5 points), 30 mg/M² (3 points), 60 mg/m² (3points), 75 mg/M² (7 points), and 90 mg/M² (3 points). No dose limitingtoxicity has been observed.

Two efficacious responses have been observed. One modestly favorableresponse was in a subject with T cell cutaneous lymphoma in a patientconsidered resistant to oral all-trans retinoic acid. This patient ispresently receiving a second 28 day treatment cycle.

A subject with APL in first relapse 10 months after receiving oralall-trans retinoic acid in three weeks of the liposomal treatment of thepresent invention displayed a rising white count and evidence ofincreased cellular differentiation in both the blood and the marrow.

Pharmacokinetic drug level data was also compared to published data forall-trans retinoic acid. As taken form Trump et al., ASCO Droc., Vol.13, page 241 (1994) referencing a with prostate cancer, all-transretinoic acid (non-liposomal) administered orally at 50 mg/M², twice perday for 14 days yielded a C_(max) in ng/ml on day 1 of 307 and day 14 of144. The AUC in μg hr/ml was 0.693 on day 1 and 0.250 on day 14. Asubstantially distinct result was obtained using the liposomal all-transretinoic acid of the present invention in one patient administered 60mg/M² every other day for 15 doses i.v. The C_(b0) (the concentration inblood at the conclusion of i.v. administration, time 0) in μg/ml was 6.8on day one and 7.0 on day 15 after the eighth dose. The AUC in μg/ml×minwas 466 on day 1 and 580 on day 15. Converting to μg hr/ml these valuesare 7.76 and 9.66 respectively.

The clearance of liposomal all-trans retinoic acid was found to closelyfit (r² >0.9) a two compartment mathematical model in 14 of 22 completeanalyses, and was best fit by a one compartment model in 8 of 22studies. Where present, alpha-phase half-lives ranged from 56±20 minutesat the 15 mg/m² dose level to 116±43 minutes at the highest levelanalyzed, 75 mg/M². There were no statistically significant differences(Student's t-test at ρ<0.05) in the calculated half lives between day 1and 15. In addition, there were no significant differences in half-livesat the different dose levels studied.

The apparent volume of distribution (V_(d)) was 25±2 liters at the 15mg/m² dose level (day 1) suggesting rapid distribution into a spaceapproximating total body water. As observed with the half-life data,there were no statistically significant differences in V_(d) betweensubjects at the different dose levels or between patients treated on day1 or 15. Both the C_(b0) and the extrapolated area under theconcentration curve (C_(xt)) were found to increase proportionately overthe dose range studied. Further, this range was not statisticallydifferent from Day 1 to 15. These pharmacokinetic studies disclose thatthe liposomal formulation of the present invention maintains blood leveland does not exhibit the reduction in blood levels(retinoid resistance),or other parameters associated with prolonged oral administration ofall-trans retinoic acid. Further, in the instant study, the absence ofdose-dependent and time-dependent increases in pharmacokineticparameters indicate no apparent saturation of drug clearance mechanisms.

The preceding description is intended to illustrate specific embodimentsof the present invention. It is not intended to be an exhaustive list ofall possible embodiments. Person skilled in the relevant field willrecognize that modifications could be made to the specific embodimentswhich have been disclosed, that would remain within the scope of theinvention.

We claim:
 1. A method of inhibiting the growth of retinoic acidresponsive cancer cells and avoiding all-trans retinoic acid resistance,said method, comprising administering to a living subject atherapeutically effective amount of a retinoic acid composition whichcomprises all-trans retinoic acid, liposomes whose lipid componentconsists essentially of dimyristoyl phosphatidyl choline, and atriglyceride; where the retinoic acid is substantially uniformlydistributed with the dimyristoyl phosphatidyl choline in the liposomes,where the molar ratio of retinoic acid to dimyristoyl phosphatidylcholine is at least about 15:85, where the triglyceride is at leastabout 15% by weight of the composition, and where the composition isstable in an aqueous environment.
 2. The composition of claim 1 whereinthe triglyceride from is soy bean oil.